The objective of this project is to study the function of the major methylated bases in DNA and RNA with special reference to the function of 5-methylcytosine in the DNA of eukaryotic cells. The general procedure is to extend a method developed in this laboratory for finding mutants of Escherichia coli which are defective in methylation of DNA or RNA to a eukaryote. Five types of E. coli mutants have been characterized: (1) mutants which are deficient in their ability to methylate DNA adenine; (2) mutants deficient in their ability to methylate DNA cytosine; (3) mutants with decreased levels of ribothymine in tRNA; (4) a mutant with decreased levels of 7-methylguanosine in tRNA; (5) a mutant whose tRNA lacks 2-thio-5-methylaminouridine.